INMUNOCAT study: The impact of molecular diagnosis on immunotherapy prescription in pollen polysensitized patients from Catalonia

Abstract Background Recognition of specific allergens triggering immune response is key for the appropriate prescription of allergen‐specific immunotherapy (SIT). This study aimed at evaluating the impact of using the commercially available microarray ImmunoCAPTM ISAC 112 (Thermo Fisher Scientific) on the etiological diagnosis and SIT prescription compared to the conventional diagnostic methods in patients with allergic rhinitis/rhinoconjunctivitis and/or asthma. Methods 300 patients with respiratory allergic disease, sensitized to three or more pollen aeroallergens from different species, as assessed by a skin prick test (SPT) and specific IgE assays (sIgE), were included in this multicentric, prospective observational study. SPT and a blood test were performed to all patients. Total serum IgE and sIgE (ImmunoCAPTM) for allergens found positive in the SPT and sIgE allergen components (ImmunoCAPTM ISAC 112) were measured. Results According to SPT results, the most prevalent pollen sensitizers in our population were Olea europaea followed by grass, Platanus acerifolia and Parietaria judaica. The molecular diagnosis (MD) revealed Ole e 1 as the most prevalent pollen sensitizer, followed by Cup a 1, Phl p 1, Cyn d 1, Par j 2, Pla a 1, 2, and 3 and Phl p 5. Immunotherapy prescription changed, due to MD testing, in 51% of the cases, with an increase of prescription of SIT from 39% to 65%. Conclusion The identification of the allergen eliciting the respiratory disease is essential for a correct immunotherapy prescription. The advances in allergen characterization using methods, such as the commercial microarray ImmunoCAPTM ISAC 112, can help clinicians to improve SIT prescription.


| INTRODUCTION
Catalonia is a region located in the northeast of the Iberian Peninsula Aeroallergy trends in a certain population reflect exposure to pollens and other aeroallergens, such as dust mites, molds or dander. 2 It has been widely documented that the prevalence of respiratory allergic diseases, such as asthma and rhinitis/rhinoconjunctivitis (RC), has increased considerably over the recent decades. 3 This increase has been associated to climate change, which is directly related to greater pollen production and allergenicity. 4 In fact, both profile complexity of IgE sensitized patients and number of polysensitized patients in our daily medical practice have increased.
Allergen-specific immunotherapy (SIT) is based on therapeutic vaccination with specific allergens and is the only specific etiologybased treatment for allergic respiratory diseases (RC, asthma). 5 The identification of the triggering allergen is essential for a correct allergy diagnosis and SIT prescription. However, commercially available allergen extracts are obtained from natural sources and contain a mix of major and minor allergens as well as non-allergenic elements.
Additionally, there is currently a lack of standardization among the different commercial sources. Therefore, the diagnosis may not be totally accurate, especially in polysensitized individuals who are increasing in numbers. 6 The Component Resolved Diagnosis (CRD) or molecular diagnosis (MD) has been introduced more than 10 years ago in clinical practice and has changed the paradigm of allergy diagnosis especially in polysensitized patients. 7,8 Though there is still space for improvement, current MD testing methods allow, in many cases, clinicians to determine the exact molecule to which the patient is genuinely sensitized. This has raised some fundamental questions regarding a proper allergy diagnosis and an accurate SIT prescription, as follows: Is it worth to use vaccine extracts that do not contain significant amounts of the molecular component driving patient sensitization? Is it effective to use vaccines against allergens that are not genuine sensitizers? Should MD be always used in patients who are candidates for vaccines? In order to address these questions, it is important to analyze the impact of MD on SIT prescription and in case considerable changes in the prescription patterns are revealed, evaluate if such changes are associated with better patient outcomes.
While some research groups have shown that MD significantly modifies SIT prescription, [9][10][11][12] more studies assessing its impact on vaccination outcomes are warranted.
This paper aims to evaluate the epidemiology of allergy sensitization in Catalonia and to assess/confirm whether MD can modify immunotherapy prescription.

| Study design and patient selection
This is a multicentric, prospective observational study that recruited a total of 300 consecutive patients from 12 different hospitals across Catalonia, who were previously diagnosed with asthma and rhinitis, according to Spanish guidelines (supplementary Table S1). This study included patients from 3 to 82 years, attending a participating outpatient clinic between 2018 and 2019. Both the collection and processing of personal data were performed in accordance with the Spanish Organic Law 3/2018 on the protection of personal data. All personal data and sample references were anonymized using independent codes maintained into a database under secure conditions.

| Participants
Inclusion criteria were as follows: -Patients diagnosed with respiratory allergic disease within the previous 2 years: intermittent or persistent, moderate to severe, rhinitis/rhinoconjunctivitis and/or mild or moderate persistent asthma, according to ARIA 13  -Patients sensitized to three or more pollen aeroallergens from different species as confirmed by SPT. Sensitization to other aeroallergens or food allergens was not considered an exclusion criterion.
-Subjects living in the same geographical region for at least 2 years; -Patients who agreed to sign an informed consent form. For patients under 18 years of age parents or guardians had to sign the informed consent.
Patients were excluded in case of uncontrolled severe asthma, severe atopic dermatitis, dermographism, or any other baseline disease for which diagnostic testing (SPT) is controversial. Patients who had received previous allergen immunotherapy, those for whom immunotherapy prescription was not indicated and those with neoplastic and/or autoimmunologic diseases were also excluded.
Three patients aged 3, 4 and 82 years were included in the study although their age was outside the classical immunotherapy age range. They were not excluded from the analysis since for the purposes of this study the recommendation for SIT was purely hypothetical and proposed on the basis of detected sensitizations.
Sample size was inferred based on the estimation that 80% of the pediatric, adolescent, and young adult population attending the allergology outpatient clinic has some type of respiratory pathology (rhinitis, RC and/or asthma). Of these, between 30% and 65% are expected to have a positive SPT to three or more allergens. 15 Based on these data, it was calculated that a minimum of 70 patients would be required to achieve sufficient statistical power.

| Variables
Clinical, demographic, and anthropometric variables for each patient participating in the study were collected by each researcher.
SPTs were performed with standardized inhalant allergen extracts used in routine testing in every hospital, including the following pollen aeroallergens 15 transfer protein (LTP) and palm tree profilin were also included in the SPT. All patients had been instructed not to take medications during the 7 days before the test.
Histamine hydrogen chloride 10 mg/mL was used as positive control and physiologic saline as negative control. All SPTs were conducted using injections in the volar surface of each forearm. SPTs were performed following EAACI recommendations. Papules were measured at 15 min. Papules were considered positive if they were greater than 7.1 mm 2 , which would equal a papule of 3 mm in diameter.
Blood samples were taken from all patients included in the study.
The following parameters were measured: total serum IgE; specific IgE to whole allergens detected by SPT (Phadia 250 Laboratory System; Thermo Fisher Scientific); IgEs to specific molecular components (microarray ImmunoCAP TM ISAC 112; Thermo Fisher Scientific, see supplementary Table S2 for complete list of components) to have a wide picture on patient sensitization profiles. A semi quantitative measurement of molecular components was performed using a Luxcan scanner according to the manufacturer's specifications. The results were determined using ISAC standardized units and considered positive if ≥ 0.3 ISU were reported.

| Recommended prescription of allergen immunotherapy
All the researchers filled out a questionnaire for each patient including the hypothetical indication and the allergen composition of the SIT that they would (or would not) recommend based on data obtained from clinical history, SPT and specific IgE results before obtaining the results of MD. After obtaining the result of Immuno-CAP TM ISAC 112, the same researcher had to fill out a second copy of the same questionnaire. Determinations were considered in agreement if indication of SIT and selected allergens were the same in the two copies of the questionnaire (i.e., before and after MD results were obtained). In both cases, ITA was recommended according to the main guidelines that were prevailing during the study period. [17][18][19]

| Statistical analysis
A descriptive analysis of the study data has been carried out through the elaboration of frequency tables for the nominal type variables and measures of central tendency and dispersion for the continuous variables. The comparison between groups was carried out using Fisher's exact test for qualitative variables. In the case of quantitative variables, the Student's t test was performed for the comparison between 2 groups and the ANOVA test for the comparison of 3 or more groups. The ISAC result was presented by subject using a heatmap (based on ISAC classes) and using boxplots by component and by age groups. SIT use before and after MD was presented using a heatmap (use vs. no use). Detailed SIT use of the specific target allergens was presented showing the percent of subjects whose SIT contained each specific allergen both before and after MD, also segmented by age. All calculations were performed using the SAS 9.4 statistical package with a significance limit of 0.05.

| Demographics
A total of 300 patients were included in this study. Table 1

| Pollen sensitization profile according to ImmunoCAP TM ISAC 112
ImmunoCAP TM ISAC 112 results showed a similar sensitization pattern to SPT ( Figure 1B

| Immunotherapy prescription
Immunotherapy prescription changed due to MD testing in 51% of the cases ( Figure 3B). The number of patients potentially benefiting

| Immunotherapy prescription by age
The change in SIT prescription followed the same trend across all

| Evaluation of the correlation between rhinoconjunctivitis/asthma severity and the sensitization intensity to specific pollen components
The correlation between ImmunoCAP TM ISAC 112 signal intensity and the severity of the asthma/rhinoconjunctivitis was evaluated considering patients with mild and moderate disease due to the low number of patients suffering from a severe pathology (not enough to reach statistically significant results). A positive correlation was considered for p values less than 0.05. A statistically significant correlation was only found for Sal k 1 (p = 0.032) in patients suffering from rhinoconjunctivitis. Regarding asthma, no correlation was found in any of the pollen components analyzed in this study (supplementary Tables S3 and S4 and table supplementary   Figure S2).

| DISCUSSION
The main objective of this study was to evaluate the impact of MD on immunotherapy prescription.

F I G U R E 2
Comparison between ImmunoCAP ® sIgE and SPT results for the most common pollen allergens. Graph highlights the frequency of negative SPT in positive ImmunoCAP ® sIgE patients. Peach LTP = lipid transfer protein. To that end, we evaluated the patients' sensitization profiles using three different methods: SPT, ImmunoCAP TM sIgE, and ImmunoCAP TM ISAC 112. According to all methods, Olea europaea was the main sensitizing pollen, although, interestingly, Olea europaea is not extensively grown in Catalonia. In general terms, the 3 tests agreed in terms of the sensitization prevalence for each pollen although some discrepancies were found (e.g., Chenopodium album judaica showed a discrepancy between SPT and whole IgE testing in more than 10% of the patients with an average value above 1 kU A /l, indicating an important degree of misdiagnosis from SPT for these particular allergens. Despite the general agreement among testing methods with respect to the main allergenic pollens, many discrepancies have been found between SPT and MD methods (see above), which are in agreement with other publications. 21,22 Therefore, MD has a significant impact on SIT prescription in our region at three different levels:

F I G U R E 3
Changes in the immunotherapy (SIT) prescriptions following molecular diagnosis. (A), Percentage of patients who were prescribed SIT (candidates for immunotherapy) versus percentage of patients that were not prescribed SIT (non-candidates for immunotherapy) before and after molecular diagnosis. (B), Changes in the SIT prescriptions following molecular diagnosis: patients who became eligible for SIT ("No to Yes": 33%), patients who were no longer considered candidates for SIT ("Yes to "No": 6%), or patients who were still eligible for SIT, but had their SIT prescription adjusted. (C and D), Adjustments in the SIT prescription in terms of the allergens (C) composition and (D) number.
changing the SIT composition, helping identify new candidates for SIT, and revealing that some patients who would have been recommended SIT based on SPT results were not good candidates after all.
In fact, SIT prescription changed due to MD for 51% of the participating patients, matching previous reported data. 10

ACKNOWLEDGMENTS
We would like to thank the patients and families who participated in this study for their contribution to scientific progress. We also wish to acknowledge the Catalan Society of Allergy and Clinical Immunology (SCAIC) for providing the ideal framework for academic research. The authors thank Dani Granados who elaborated the online database. We also thank Thermo Fisher Scientific for performing the statistical analysis and for reviewing the English version of this manuscript. Thermo Fisher Scientific, Allergy-Therapeutics, Alk-Abelló, Diater, Hal-Allergy, Leti, Roxall Group, and Stallergenes Greer